CN108277266A - Detect method and reagent of the archaeal dna polymerase to the fault-tolerant ability of the base mismatch formed in sequencing procedure - Google Patents
Detect method and reagent of the archaeal dna polymerase to the fault-tolerant ability of the base mismatch formed in sequencing procedure Download PDFInfo
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Abstract
Method and reagent of the archaeal dna polymerase to the fault-tolerant ability of the base mismatch formed in sequencing procedure are detected the invention discloses a kind of, the method includes：At least one chip and an at least mismatched primers for being mounted with nucleic acid is provided, the mismatched primers at least number of base in its 3 ' last position and nucleic acid mismatch, other parts is correctly matched with nucleic acid, so that mismatched primers can be extended by template of nucleic acid；Next base complementrity of at least one archaeal dna polymerase to be measured and at least one dNTP with fluorescence, the dNTP with fluorescence and 3 ' the corresponding bases on nucleic acid in last position are provided；The chip for being mounted with nucleic acid, mismatched primers, archaeal dna polymerase to be measured and dNTP with fluorescence are incubated under conditions of suitable for primer extend, and the fluorescence signal for being incubated product is acquired, fault-tolerant ability of the archaeal dna polymerase to be measured to the base mismatch formed in sequencing procedure is calculated according to fluorescence signal.When the method for the present invention can detect mispairing and generate, polymerizing power of the archaeal dna polymerase to next bit base after mispairing.
Description
Technical field
The present invention relates to archaeal dna polymerase performance study technical fields more particularly to a kind of detection archaeal dna polymerase to being sequenced
The method and reagent of the fault-tolerant ability of the base mismatch formed in journey.
Background technology
Archaeal dna polymerase participates in the duplication of DNA molecular.When archaeal dna polymerase synthesizes nascent strand, need with DNA to be to replicate template,
And primer is needed to originate synthesis, (dATP, dCTP, dGTP and dTTP are referred to as four kinds of deoxynucleoside triphosphates of catalysis
DNTPs), along the new DNA chain of 5 ' to 3 ' direction compositions.In the sequencing of DNA (Sanger, et.al.,
Proc.Natl.Acad.Sci.,USA 74:5463-5467 (1977)), archaeal dna polymerase has key effect, to inhomogeneity
The polymerizing power of type dNTPs and the quality of sequencing are closely related.
It is sequenced in (SBS) technology existing when synthesizing, the archaeal dna polymerase used can be added with fluorescent marker
dNTPs(Prober,et.al.,Science 238:336-341 (1987)), consequently facilitating reading DNA sequence dna information.But it is surveying
In program process, the false bases that are added once in a while the addition of next bit base can be made to be obstructed so that influence the continuation of sequencing reaction into
Row so that sequencing signal significantly declines, and causes sequencing quality relatively low.
DNTPs the or dNTP analogs with modification and blocking group can be added in archaeal dna polymerase used at present, but existing
There is technology that can not judge whether archaeal dna polymerase can also continuously add next bit alkali in the case where false bases have been added to
Base, can not also analyze next bit base addition efficiency how.
Invention content
The present invention provides a kind of method detecting archaeal dna polymerase to the fault-tolerant ability of the base mismatch formed in sequencing procedure
And reagent, when can detect mispairing and having generated, polymerizing power of the archaeal dna polymerase to next bit base after mispairing.
According to the first aspect of the invention, the present invention provides a kind of detection archaeal dna polymerase to the mistake that is formed in sequencing procedure
The method of fault-tolerant ability with base, including：
(a) at least one chip and an at least mismatched primers for being mounted with nucleic acid be provided, the mismatched primers in its 3 ' last position and
Above-mentioned nucleic acid mismatch, at least number of base is correctly matched with above-mentioned nucleic acid in other parts, so that above-mentioned mismatched primers can
Extended as template using above-mentioned nucleic acid；
(b) provide at least one archaeal dna polymerase to be measured and at least one dNTP with fluorescence, the above-mentioned dNTP with fluorescence with it is above-mentioned
3 ' last positions correspond to next base complementrity of the base on above-mentioned nucleic acid；
(c) by the above-mentioned chip for being mounted with nucleic acid, above-mentioned mismatched primers, above-mentioned archaeal dna polymerase to be measured and above-mentioned band fluorescence
DNTP be incubated under conditions of suitable for primer extend, and acquire be incubated product fluorescence signal, according to above-mentioned fluorescence believe
Number calculate the fault-tolerant ability of above-mentioned archaeal dna polymerase to be measured to the base mismatch formed in above-mentioned sequencing procedure.
In the above method, step (a) and step (b) can first carry out one of step without sequencing, can also be same
Two steps of Shi Jinhang.
Further, above-mentioned mismatched primers in its 3 ' last position with above-mentioned nucleic acid mismatch and close to the upstream alkaline of above-mentioned 3 ' last positions
Base is correctly matched with above-mentioned nucleic acid, and at least number of base is correctly matched with above-mentioned nucleic acid in other parts.
Further, above-mentioned mismatched primers are in addition to several with above-mentioned nucleic acid mismatch and in its 5 ' end permission in its 3 ' last position
Other than mispairing, other parts are correctly matched with above-mentioned nucleic acid completely.
Further, for above-mentioned mismatched primers other than in its 3 ' last position and above-mentioned nucleic acid mismatch, other parts are complete
It is correctly matched with above-mentioned nucleic acid.
Further, the above method further includes：
(d) an at least normal primer is provided, which correctly matches in its 3 ' last position with above-mentioned nucleic acid, so as to above-mentioned
Normal primer can be extended using above-mentioned nucleic acid as template；
(e) by the above-mentioned chip for being mounted with nucleic acid, above-mentioned normal primer, above-mentioned archaeal dna polymerase to be measured and above-mentioned band fluorescence
DNTP be incubated under conditions of suitable for primer extend, and acquire be incubated product fluorescence signal, with the fluorescence signal make
Judge whether the fluorescence signal of above-mentioned (c) step meets above-mentioned standard fluorescence signal for standard fluorescence signal.
In the above method, step (a) to step (c) as a whole with step (d) to step (e) as a whole
Without sequencing, it can first carry out (a) and carry out step (d) to step (c) or first to step (e), work can also be carried out at the same time
For an entirety step (a) to step (c) with as a whole the step of (d) to step (e).
Further, above-mentioned normal primer in its 3 ' last position with above-mentioned nucleic acid in addition to correctly matching and allowing at its 5 ' end
Other than several mispairing, other parts are correctly matched with above-mentioned nucleic acid completely.
Further, above-mentioned normal primer is correctly matched with above-mentioned nucleic acid completely.
Further, above-mentioned nucleic acid is DNA nanospheres.
Further, above-mentioned standard fluorescence signal is the fluorescence signal within the scope of predetermined fluorescent intensity, correspondingly, more than
State (c) step fluorescence signal fall within the scope of above-mentioned predetermined fluorescent intensity as can be fault-tolerant index, count above-mentioned (c) step
Reaction that can be fault-tolerant in rapid multiple parallel reactions accounts for the percentage of overall reaction number；
Preferably, above-mentioned predetermined fluorescent intensity range refers to 75% ± 2.5% fluorescence intensity range.
Further, above-mentioned fluorescence signal is by choosing the block of predetermined number on chip, to base and corresponding glimmering
Optical signal value is analyzed, it is preferable that above-mentioned predetermined number is 5.
According to the second aspect of the invention, the present invention provides a kind of detection archaeal dna polymerase to the mistake that is formed in sequencing procedure
The reagent of fault-tolerant ability with base, including：
(a) at least one chip and an at least mismatched primers for being mounted with nucleic acid, the mismatched primers its 3 ' last position with it is above-mentioned
Nucleic acid mismatch, at least number of base is correctly matched with above-mentioned nucleic acid in other parts, so as to above-mentioned mismatched primers can more than
It is that template is extended to state nucleic acid；
(b) at least one archaeal dna polymerase to be measured and at least one dNTP with fluorescence, the above-mentioned dNTP with fluorescence and above-mentioned 3 ' end
Next base complementrity of the corresponding base on above-mentioned nucleic acid in position；
(c) the above-mentioned chip for being mounted with nucleic acid, above-mentioned mismatched primers, above-mentioned archaeal dna polymerase to be measured and above-mentioned with fluorescence
DNTP for being incubated under conditions of suitable for primer extend, and acquires the fluorescence signal for being incubated product, according to above-mentioned fluorescence
Signal calculates fault-tolerant ability of the above-mentioned archaeal dna polymerase to be measured to the base mismatch formed in above-mentioned sequencing procedure.
The method and reagent of the present invention, can detect in the case where mispairing has occurred for base, archaeal dna polymerase continuously adds down
The ability and efficiency of one bit base can be used for researching and developing and optimize the archaeal dna polymerase used in high-flux sequence.The detection method
It can be used for all types of archaeal dna polymerases, can be used for proofreading and the repair ability of detection archaeal dna polymerase.
Description of the drawings
Fig. 1 is that mismatched primers form pairing with nucleic acid and the signal of next base is added in one embodiment of the invention
Figure；
Fig. 2 is the schematic diagram of design of primers and illustrative primer in one embodiment of the invention.
Specific implementation mode
Below by specific implementation mode combination attached drawing, invention is further described in detail.
In an embodiment of the invention, as shown in Figure 1, upper Sorted list indicates normal primer (the case where 3 ' last positions are C
Under) and mismatched primers (in the case that 3 ' last positions replace with A, G or T), lower sorted lists are shown as the nucleic acid sequence of template；According to
Nucleic acid sequence as template is it is found that the next base to be added is A.
It should be noted that Fig. 1 is only an illustrative example, in the different location as the nucleic acid sequence of template
Different normal primer and mismatched primers can be designed, such as the base of the last position of normal primer 3 ' can be A, G or T successively,
In the case of, the base of the last position of mismatched primers 3 ' could alternatively be other three kinds of bases, such as the last position of normal primer 3 '
In the case that base is A, the base of the last position of mismatched primers 3 ' replaces with C, G or T；The feelings that the base of the last position of normal primer 3 ' is G
Under condition, the base of the last position of mismatched primers 3 ' replaces with C, A or T；In the case that the base of the last position of normal primer 3 ' is T, mispairing is drawn
The base of the last position of object 3 ' replaces with C, G or A.Meanwhile at each occurrence, the next base to be added can be A, G, C
Or any one in T.
Fig. 2 shows the schematic diagrames of design of primers and illustrative primer in one embodiment of the invention.It can from Fig. 2
Going out, the base of 3 ' last positions can be any one in A, G, C or T, correspondingly, any one in A, G, C or T is could alternatively be,
The next base to be added can be any one in A, G, C or T simultaneously, and such one shares 4 × 4 × 4=64 kind primer sequences
Row, including 48 kinds of 16 kinds of normal primer and mismatched primers.Therefore, by using the method for these primers and the present invention, energy
After enough detecting any one base mispairing, archaeal dna polymerase is normally added the ability of next base, that is, formed base mismatch with
Fault-tolerant ability afterwards.
Fig. 2 also shows the example of illustrative four primers, and the base with 3 ' last positions is A's (base of overstriking in figure)
Primer replaces with the primer of G, C or T as mismatched primers respectively as normal primer, using the base of 3 ' last positions, and this four
The primer next base to be added is A (base in figure center).
The mismatched primers of the embodiment of the present invention its 3 ' last position with as the nucleic acid sequence mispairing of template, in other parts extremely
Rare number of base is correctly matched with the nucleic acid sequence as template, so as to the mismatched primers can using nucleic acid sequence as template into
Row extends.That is, other than 3 ' last positions are with nucleic acid sequence mispairing, the base of mismatched primers other parts does not require completely
It is correctly matched with nucleic acid sequence, as long as the mismatched primers can be extended by template of nucleic acid sequence.
However, from the purpose for realizing reliable effect, close to 3 ' last positions upstream base (i.e. on 3 ' to 5 ' directions,
Close to the base of 3 ' last positions) should correctly be matched with the nucleic acid sequence as template, avoid in this way 3 ' ends there are two with
The case where upper continuous base mismatch.That is, in a preferred embodiment of the invention, mismatched primers are in its 3 ' last position
Upstream base with the nucleic acid sequence mispairing as template and close to 3 ' last positions is correctly matched with the nucleic acid sequence as template,
At least number of base is correctly matched with nucleic acid sequence in its part.
From the purpose for realizing more preferably effect, mismatched primers are in addition to wrong in its 3 ' last position and the nucleic acid sequence as template
Match, and allow other than several bases (such as 1-5,6 or 8 bases) mispairing at its 5 ' end, other parts are completely and nucleic acid
Sequence is correctly matched.It can ensure that the pairing of other parts is continuous other than several mispairing at 3 ' last positions and 5 ' ends in this way
, to ensure that extension more efficiently carries out.
From the purpose for realizing optimum efficiency, mismatched primers are in addition to wrong in its 3 ' last position and the nucleic acid sequence as template
With in addition, other parts are correctly matched with nucleic acid sequence completely.
The normal primer of the embodiment of the present invention is correctly matched in its 3 ' last position with the nucleic acid sequence as template, so as to normal
Primer can be extended by template of nucleic acid sequence.Similar to mismatched primers, normal primer is in addition to 3 ' last positions and as template
Nucleic acid sequence correctly pairing other than, the base of normal primer other parts does not require correctly to match with nucleic acid sequence completely, only
Will the normal primer can be extended by template of nucleic acid sequence.
From the purpose for realizing more preferably effect, normal primer in addition to its 3 ' last position with the nucleic acid sequence as template just
Really pairing, and allowing other than several bases (such as 1-5,6 or 8 bases) mispairing at its 5 ' end, other parts completely with
Nucleic acid sequence is correctly matched.It can ensure that the pairing of other parts is continuous other than several mispairing at 5 ' ends in this way, from
And ensures extension and more efficiently carry out.
From the purpose for realizing optimum efficiency, normal primer is correctly matched with the nucleic acid sequence as template completely.
In the embodiment of the present invention, archaeal dna polymerase to be measured can be any archaeal dna polymerase used in sequencing procedure.This hair
In bright embodiment, the dNTP with fluorescence can be any dNTP with fluorescence used in sequencing procedure, such as be surveyed in synthesis
The dNTP with fluorescence used in sequence (SBS) technology, as long as 3 ' the last positions of the dNTP with fluorescence and normal primer and mismatched primers
Base-pair should be in next base complementrity of the base in the nucleic acid sequence as template, such as arrow is signified in Fig. 1
The A shown is the dNTP with fluorescence.
Principle of the present invention using the dNTP with fluorescence as reaction substrate is will to be mounted with the chip of nucleic acid, mispairing is drawn
Object, archaeal dna polymerase to be measured and the dNTP with fluorescence are incubated under conditions of suitable for primer extend, if the dNTP with fluorescence
The position of next base can be added, then be able to detect that scheduled fluorescence signal；If the dNTP with fluorescence cannot be added
The position of next base cannot then detect scheduled fluorescence signal.Therefore, can be speculated according to the fluorescence signal detected
Fault-tolerant ability of the archaeal dna polymerase to be measured to the base mismatch formed in sequencing procedure.
Above-mentioned " scheduled fluorescence signal " can use normal primer as the collected fluorescence signal of extension primer,
Such as the chip of nucleic acid, normal primer, archaeal dna polymerase to be measured and dNTP with fluorescence will be mounted in the item suitable for primer extend
It is incubated under part, and acquires the fluorescence signal for being incubated product, also referred to as standard fluorescence signal.
In one embodiment of the invention, it is DNA nanospheres (DNB), such as side synthesis as the nucleic acid sequence of template
The DNB used in side sequencing (SBS) technology.In this case, normal primer and mismatched primers guiding extension into
The multiple parallel repetition reactions of row, the reaction site on chip where each corresponding DNB of reaction.In this way, " scheduled glimmering
Optical signal " can be the fluorescence letter that the fluorescence signal of the extension of normal primer guiding is fallen into " predetermined fluorescent intensity range "
Number, wherein the fluorescence that multiple extensions that the fluorescence signal of " predetermined fluorescent intensity range " can be normal primer guiding obtain
The average value of signal can also be such as in an embodiment of the invention, to refer between a certain range of fluorescence signal
Fluorescence signal within the scope of 75% ± 2.5% fluorescence intensity, 75% ± 2.5% indicates that multiple extensions of normal primer guiding are anti-
In the fluorescence signal that should be obtained, intensity between 75% ± 2.5% fluorescence signal.Therefore, in the embodiment of the present invention, fault-tolerant ability
Detection be converted to analysis mismatched primers guiding multiple (such as 100 or 1000 etc.) extensions obtain fluorescence signal
The reaction number fallen into " predetermined fluorescent intensity range " (such as 75% ± 2.5%) accounts for the percentage of overall reaction number.
In the case where using nucleic acid sequences of the DNB as template, fluorescence signal is by choosing predetermined number on chip
Block (Block) analyzes base and corresponding fluorescence signal value, it is preferable that above-mentioned predetermined number is 5.Due to sample
This amount is few, therefore analyze speed is very fast, and by choosing specific chip block, can reflect the sequencing feelings of whole chip
Condition.
The technical solution that the present invention will be described in detail by the following examples, it should be understood that embodiment is merely exemplary, no
It can be interpreted as limiting the scope of the invention.
Embodiment 1
For using BGISEQ-1000 microarray datasets and AD153 connectors as the nucleic acid sequence of template, design last position introduce it is wrong
The primer matched, and detect in the case where mispairing has occurred for base, archaeal dna polymerase continuously adds the ability and effect of next bit base
Rate.
By taking the mispairing (dATP is replaced by dGTP, dCTP or dTTP respectively) of dATP as an example, if next bit base is dGTP,
The normal primer used and mismatched primers are as shown in table 1.
Table 1
Experiment condition：As shown in table 2, wherein L01 and L08 is normal sequencing reaction, and different experiments is used in other L
Condition.
Table 2
As a result：DATP is replaced by dGTP, dCTP or dTTP after mispairing, and the addition rate of next bit dGTP is different, with
The specific addition rate of 75% ± 2.5% signal strength analysis, next bit dGTP is as shown in table 3 below.
Table 3
Embodiment 2
The present embodiment devises 64 kinds of primers (being shown in Table 4), and is carried out according to the similar reaction of embodiment 1, with 75% ±
In the case of 2.5% signal strength analysis, various mispairing and various next bit bases, specific addition rate is as shown in table 5 below.
Table 4
Note：By taking TxGnC as an example：1st T represents original base, and the 3rd G represents the base after mispairing, and the 5th C is represented
Next bit base of the chain of script；X represents " being replaced by "；N represents " next bit (next) "；Base expression in bracket will add
The next bit base entered.
Table 5
The above content is combining, specific embodiment is made for the present invention to be further described, and it cannot be said that this hair
Bright specific implementation is confined to these explanations.For those of ordinary skill in the art to which the present invention belongs, it is not taking off
Under the premise of from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to the protection of the present invention
Range.
SEQUENCE LISTING
<110>BGI-Shenzhen
<120>Detect method and reagent of the archaeal dna polymerase to the fault-tolerant ability of the base mismatch formed in sequencing procedure
<130> P2016-1-0143.CN
<160> 69
<170> PatentIn version 3.3
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<213>Primer sequence
<400> 33
ctcctaacga cacagtggct catgt 25
<210> 34
<211> 25
<212> DNA
<213>Primer sequence
<220>
<221> misc_feature
<222> (1)..(7)
<223> n is a, c, g, or t
<400> 34
nnnnnnncca agggaggaag tccgg 25
<210> 35
<211> 25
<212> DNA
<213>Primer sequence
<220>
<221> misc_feature
<222> (1)..(7)
<223> n is a, c, g, or t
<400> 35
nnnnnnncca agggaggaag tccga 25
<210> 36
<211> 25
<212> DNA
<213>Primer sequence
<220>
<221> misc_feature
<222> (1)..(7)
<223> n is a, c, g, or t
<400> 36
nnnnnnncca agggaggaag tccgc 25
<210> 37
<211> 25
<212> DNA
<213>Primer sequence
<220>
<221> misc_feature
<222> (1)..(7)
<223> n is a, c, g, or t
<400> 37
nnnnnnncca agggaggaag tccgt 25
<210> 38
<211> 25
<212> DNA
<213>Primer sequence
<400> 38
cggtcccaag ggaggttagg aagac 25
<210> 39
<211> 25
<212> DNA
<213>Primer sequence
<400> 39
cggtcccaag ggaggttagg aagaa 25
<210> 40
<211> 25
<212> DNA
<213>Primer sequence
<400> 40
cggtcccaag ggaggttagg aagag 25
<210> 41
<211> 25
<212> DNA
<213>Primer sequence
<400> 41
cggtcccaag ggaggttagg aagat 25
<210> 42
<211> 26
<212> DNA
<213>Primer sequence
<220>
<221> misc_feature
<222> (1)..(5)
<223> n is a, c, g, or t
<400> 42
nnnnncaaca cagtggctct cctaac 26
<210> 43
<211> 26
<212> DNA
<213>Primer sequence
<220>
<221> misc_feature
<222> (1)..(5)
<223> n is a, c, g, or t
<400> 43
nnnnncaaca cagtggctct cctaaa 26
<210> 44
<211> 26
<212> DNA
<213>Primer sequence
<220>
<221> misc_feature
<222> (1)..(5)
<223> n is a, c, g, or t
<400> 44
nnnnncaaca cagtggctct cctaag 26
<210> 45
<211> 26
<212> DNA
<213>Primer sequence
<220>
<221> misc_feature
<222> (1)..(5)
<223> n is a, c, g, or t
<400> 45
nnnnncaaca cagtggctct cctaat 26
<210> 46
<211> 25
<212> DNA
<213>Primer sequence
<400> 46
ctctcctaac gacatggcta cgatc 25
<210> 47
<211> 25
<212> DNA
<213>Primer sequence
<400> 47
ctctcctaac gacatggcta cgata 25
<210> 48
<211> 25
<212> DNA
<213>Primer sequence
<400> 48
ctctcctaac gacatggcta cgatg 25
<210> 49
<211> 25
<212> DNA
<213>Primer sequence
<400> 49
ctctcctaac gacatggcta cgatt 25
<210> 50
<211> 25
<212> DNA
<213>Primer sequence
<400> 50
tccttggctc tcctaacgac atggc 25
<210> 51
<211> 25
<212> DNA
<213>Primer sequence
<400> 51
tccttggctc tcctaacgac atgga 25
<210> 52
<211> 25
<212> DNA
<213>Primer sequence
<400> 52
tccttggctc tcctaacgac atggg 25
<210> 53
<211> 25
<212> DNA
<213>Primer sequence
<400> 53
tccttggctc tcctaacgac atggt 25
<210> 54
<211> 25
<212> DNA
<213>Primer sequence
<400> 54
ccttggctct cctaacgaca tggct 25
<210> 55
<211> 25
<212> DNA
<213>Primer sequence
<400> 55
ccttggctct cctaacgaca tggca 25
<210> 56
<211> 25
<212> DNA
<213>Primer sequence
<400> 56
ccttggctct cctaacgaca tggcg 25
<210> 57
<211> 25
<212> DNA
<213>Primer sequence
<400> 57
ccttggctct cctaacgaca tggcc 25
<210> 58
<211> 26
<212> DNA
<213>Primer sequence
<400> 58
caactccttg gctctcctaa cgacat 26
<210> 59
<211> 26
<212> DNA
<213>Primer sequence
<400> 59
caactccttg gctctcctaa cgacaa 26
<210> 60
<211> 26
<212> DNA
<213>Primer sequence
<400> 60
caactccttg gctctcctaa cgacag 26
<210> 61
<211> 26
<212> DNA
<213>Primer sequence
<400> 61
caactccttg gctctcctaa cgacac 26
<210> 62
<211> 25
<212> DNA
<213>Primer sequence
<400> 62
gctctcctaa cgacatggct acgat 25
<210> 63
<211> 25
<212> DNA
<213>Primer sequence
<400> 63
gctctcctaa cgacatggct acgaa 25
<210> 64
<211> 25
<212> DNA
<213>Primer sequence
<400> 64
gctctcctaa cgacatggct acgag 25
<210> 65
<211> 25
<212> DNA
<213>Primer sequence
<400> 65
gctctcctaa cgacatggct acgac 25
<210> 66
<211> 25
<212> DNA
<213>Primer sequence
<400> 66
agaacgaatc cgctacgcat ggact 25
<210> 67
<211> 25
<212> DNA
<213>Primer sequence
<400> 67
agaacgaatc cgctacgcat ggaca 25
<210> 68
<211> 25
<212> DNA
<213>Primer sequence
<400> 68
agaacgaatc cgctacgcat ggacg 25
<210> 69
<211> 25
<212> DNA
<213>Primer sequence
<400> 69
agaacgaatc cgctacgcat ggacc 25
Claims (10)
1. a kind of detection archaeal dna polymerase is to the method for the fault-tolerant ability of the base mismatch formed in sequencing procedure, which is characterized in that
The method includes：
(a) at least one chip and an at least mismatched primers for being mounted with nucleic acid be provided, the mismatched primers its 3 ' end position with it is described
Nucleic acid mismatch, at least number of base is correctly matched with the nucleic acid in other parts, so that the mismatched primers can be with institute
It is that template is extended to state nucleic acid；
(b) at least one archaeal dna polymerase to be measured and at least one dNTP with fluorescence, the dNTP with fluorescence and the 3 ' end are provided
Next base complementrity of the corresponding base on the nucleic acid in position；
(c) by the chip for being mounted with nucleic acid, the mismatched primers, the archaeal dna polymerase to be measured and described with fluorescence
DNTP is incubated under conditions of suitable for primer extend, and acquires the fluorescence signal for being incubated product, according to the fluorescence signal
Calculate fault-tolerant ability of the archaeal dna polymerase to be measured to the base mismatch formed in the sequencing procedure.
2. detection archaeal dna polymerase according to claim 1 is to the fault-tolerant ability of the base mismatch formed in sequencing procedure
Method, which is characterized in that the mismatched primers are in its 3 ' last position with the nucleic acid mismatch and close to the upstream alkaline of described 3 ' last positions
Base is correctly matched with the nucleic acid, and at least number of base is correctly matched with the nucleic acid in other parts.
3. detection archaeal dna polymerase according to claim 2 is to the fault-tolerant ability of the base mismatch formed in sequencing procedure
Method, which is characterized in that the mismatched primers in its 3 ' last position and the nucleic acid mismatch and at its 5 ' end in addition to allowing several mistakes
With in addition, other parts are correctly matched with the nucleic acid completely.
4. detection archaeal dna polymerase according to claim 3 is to the fault-tolerant ability of the base mismatch formed in sequencing procedure
Method, which is characterized in that the mismatched primers in addition in its 3 ' last position with the nucleic acid mismatch other than, other parts are completely and institute
Nucleic acid is stated correctly to match.
5. detecting appearance of the archaeal dna polymerase to the base mismatch formed in sequencing procedure according to claim 1-4 any one of them
The method of wrong ability, which is characterized in that the method further includes：
(d) an at least normal primer is provided, which correctly matches in its 3 ' last position with the nucleic acid, so as to described normal
Primer can be extended using the nucleic acid as template；
(e) by the chip for being mounted with nucleic acid, the normal primer, the archaeal dna polymerase to be measured and described with fluorescence
DNTP is incubated under conditions of suitable for primer extend, and acquire be incubated product fluorescence signal, using the fluorescence signal as
Standard fluorescence signal judges whether the fluorescence signal of described (c) step meets the standard fluorescence signal.
6. detection archaeal dna polymerase according to claim 5 is to the fault-tolerant ability of the base mismatch formed in sequencing procedure
Method, which is characterized in that if the normal primer in its 3 ' last position with the nucleic acid in addition to correctly matching and allowing at its 5 ' end
Other than dry mispairing, other parts are correctly matched with the nucleic acid completely；
Preferably, the normal primer is correctly matched with the nucleic acid completely.
7. detection archaeal dna polymerase according to claim 5 is to the fault-tolerant ability of the base mismatch formed in sequencing procedure
Method, which is characterized in that the nucleic acid is DNA nanospheres.
8. detection archaeal dna polymerase according to claim 7 is to the fault-tolerant ability of the base mismatch formed in sequencing procedure
Method, which is characterized in that the standard fluorescence signal is the fluorescence signal within the scope of predetermined fluorescent intensity, correspondingly, with described
(c) fluorescence signal of step fall within the scope of the predetermined fluorescent intensity as can be fault-tolerant index, (c) step described in statistics
Multiple parallel reactions in can be fault-tolerant reaction account for the percentage of overall reaction number；
Preferably, the predetermined fluorescent intensity range refers to 75% ± 2.5% fluorescence intensity range.
9. detection archaeal dna polymerase according to claim 8 is to the fault-tolerant ability of the base mismatch formed in sequencing procedure
Method, which is characterized in that the fluorescence signal believes base and corresponding fluorescence by the block of predetermined number on selection chip
Number value is analyzed, it is preferable that the predetermined number is 5.
10. a kind of detection archaeal dna polymerase exists to the reagent of the fault-tolerant ability of the base mismatch formed in sequencing procedure, feature
In the reagent includes：
(a) at least one chip and an at least mismatched primers for being mounted with nucleic acid, the mismatched primers are in its 3 ' last position and the nucleic acid
Mispairing, at least number of base is correctly matched with the nucleic acid in other parts, so that the mismatched primers can be with the core
Acid is that template is extended；
(b) at least one archaeal dna polymerase to be measured and at least one dNTP with fluorescence, the dNTP with fluorescence and described 3 ' last positions are right
It should be in next base complementrity of the base on the nucleic acid；
(c) chip, the mismatched primers, the archaeal dna polymerase to be measured and the dNTP with fluorescence for being mounted with nucleic acid,
For being incubated under conditions of suitable for primer extend, and the fluorescence signal for being incubated product is acquired, according to the fluorescence signal
Calculate fault-tolerant ability of the archaeal dna polymerase to be measured to the base mismatch formed in the sequencing procedure.
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Citations (1)
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| CN103025868A (en) * | 2010-06-18 | 2013-04-03 | 霍夫曼-拉罗奇有限公司 | DNA polymerases with increased 3'-mismatch discrimination |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103025868A (en) * | 2010-06-18 | 2013-04-03 | 霍夫曼-拉罗奇有限公司 | DNA polymerases with increased 3'-mismatch discrimination |
Non-Patent Citations (3)
| Title |
|---|
| SRINIVAS AYYADEVARA，JOHN J. THADEN，ROBERT J. SHMOOKLER REIS: "Discrimination of Primer 39-Nucleotide Mismatch by Taq DNA Polymerase during Polymerase Chain Reaction", 《ANALYTICAL BIOCHEMISTRY》 * |
| 张明龙，张琼妮: "《美国纳米技术创新进展》", 30 June 2013, 知识产权出版社 * |
| 郭紫芬，张 佳，张 旭，廖端芳: "末端标记引物延伸反应对核酸单碱基辨认的实验研究", 《南华大学学报》 * |
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